Human herpesvirus 8 (HHV-8) is associated with endothelial Kaposi's sarcoma (KS) and B cell malignancies primary effusion lymphoma and multicentric Castleman's disease, all of which occur predominantly in the context of HIV coinfection. Both latent and lytic gene products are believed to contribute to these pathologies, and productive replication and increased viral loads are associated with KS. Understanding the processes required for successful lytic replication will provide opportunities for targeting essential viral functions to block virus replication and thereby treat or prevent HHV-8 associated disease. Key to replication by HHV-8 and other viruses are viral mechanisms that limit stress-induced apoptotic signaling that serves as an innate defense mechanism against virus infection. HHV-8 encodes several proteins that inhibit such pro-apoptotic pathways. These proteins include viral interferon regulatory factor-1 (vIRF-1), viral G protein coupled receptor (vGPCR), and viral chemokines vCCL-1 and vCCL-2, all of which have been demonstrated to both inhibit apoptosis and be required for efficient virus replication. This laboratory has identified inhibitor interactions of vIRF-1 with replication-induced pro-apoptotic BH3-only proteins (BOPs) Bim and Bid and revealed the critical importance of controlling these proteins for efficient virus replicaton to occur. Suppression of Bim or Bid expression via transduced shRNAs leads to very significant increases in virus replication, and disruption of vIRF-1:BOP interactions inhibits virus production and promotes apoptosis in lytically infected cells. In addition to Bim and Bid, vIRF-1 targets other BOPs (Bik, Bmf, Hrk, Noxa), via their functional BH3 domains, indicating the biological importance of these BOPs also and the need to inhibit their activities during lytic replication. Furthermore, the viral chemokine receptor (vGPCR) and CCR8 agonists vCCL-1 and vCCL-2 activate signaling pathways that lead to suppression of Bim expression. Thus, chemokine receptor signaling is likely to enhance HHV-8 productive replication in part via BOP regulation. This application proposes to: (1) elucidate the mechanisms of vGPCR and vCCL suppression of Bim, an identified major inhibitor of HHV-8 replication; (2) identify peptide and pharmacological inhibitors of vIRF-1:BOP interactions to specifically inhibit this mechanism of BOP regulation; (3) determine the antiviral activities of lytic cycle-induced BOPs and the individual and combined contributions, via BOP control, of vCCLs, vGPCR, and vIRF-1 to HHV- 8 productive replication. The project comprises a focused analysis of BOP function and targeting by vCCLs, vGPCR and vIRF-1 during HHV-8 lytic replication and assessment of inhibitory reagents and methods that could potentially be developed for antiviral purposes.